Peter Huy

Addressing Protein–Protein Interactions with Small Molecules: A Pro-Pro Dipeptide Mimic with a PPII Helix Conformation as a Module for the Synthesis of PRD-Binding Ligands†

Interactions of so-called proline-rich motif-recognizing domains (PRDs) with proteins containing proline-rich motifs (PRMs) are widely utilized by nature and are involved in several relevant processes, such as tyrosine kinase receptor signaling, endocytosis, cytoskeletal rearrangements, 6] transcription, and splicing. 9] In recent years, some PRDs were identified as putative therapeutical targets that can possibly be addressed by synthetic small molecules. An example is the Fyn-SH3 domain, which is involved in the regulation of enzymatic activity and the assembly of signaling complexes. A common property of all PRMs is that they preferentially form a left-handed polyproline type II (PPII) helix with an overall shape resembling a triangular prism (Figure 1). This structural element has a helical pitch of 9.3 , three residues per turn, and typical torsion angles F of 758 and Y of 1458. Of note, the PPII helix is characterized by a complete lack of main-chain hydrogen bonding patterns. Structural studies have shown that the recognition of PRMs by the respective domains (PRDs) requires the polyproline region to adopt a PPII conformation. Moreover, the interaction is mostly stabilized by formation of a hydrogen bond between a conserved tryptophane (located at the domain surface) and a carbonyl oxygen at the backbone of the central PRM of the peptide ligand. In the course of our research into the development of small molecules that specifically interfere with intracellular protein–protein interactions, we searched for a conformationally defined diproline mimic in a PPII helix conformation that could be incorporated into peptide chains or related modular constructs. To the best of our knowledge, related attempts in other laboratories have not led to the development of systems with satisfying properties to date. By investigating molecular models, we envisioned that the PPII helix could possibly be stabilized by an appropriate C2 bridge between two adjacent proline residues. Computeraided modeling then suggested that the diproline analogue X, generated by formal introduction of a “rigid” vinylidene bridge, adopts a single preferred conformation that almost perfectly mimics a Pro-Pro unit of a PPII helix (Figure 2). Herein we describe the stereoselective synthesis of this novel compound (as a Fmoc-protected derivative) and its successful incorporation as a Pro-Pro substitute in the core motif of SH3 domain-binding peptides. Figure 1. An ideal PPII helix formed by four l-proline units as seen from the side (left) and along the helix axis (right). C cyan, N blue, O red.

A modular toolkit to inhibit proline-rich motif–mediated protein–protein interactions

Significance Protein–protein interactions mediated by proline-rich motifs are involved in regulation of many important signaling cascades. Protein domains specialized in recognition of these motifs expose a flat and relatively rigid binding site that preferentially interacts with sequences adopting a left-handed polyproline helix II. Here, we present a toolkit of new chemical entities that enables rational construction of selective small-molecule inhibitors for these protein domains. As proof of principle, we developed a selective, cell-permeable inhibitor of Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains of the Ena/VASP protein family. Invasive breast-cancer cells treated with our EVH1 inhibitor showed strongly reduced cell invasion. Small-molecule competitors of protein–protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.

A practical synthesis of trans-3-substituted proline derivatives through 1,4-addition.

A practical four-step synthesis of 3-alkyl-, vinyl-, and aryl-substituted proline derivatives, which are important building blocks for conformationally restrained peptide analogs, was developed. The method relies on a Cu-catalyzed 1,4-addition of Grignard reagents to N-protected 2,3-dehydroproline esters, efficiently prepared in a new one-pot protocol. The 1,4-addition products are obtained with good trans-selectivity (dr 5:1 to 25:1). A nonracemic sample of N-Cbz-3-vinylproline (74% ee) was obtained using Evans oxazolidinone as a chiral auxiliary.

Exercises in Pyrrolidine Chemistry: Gram Scale Synthesis of a Pro–Pro Dipeptide Mimetic with a Polyproline Type II Helix Conformation

A practical and scalable synthesis of a Fmoc-protected tricyclic dipeptide mimetic (6), that is, a 1,4-diaza-tricyclo-[8.3.0(3,7)]-tridec-8-ene derivative resembling a rigidified di-L-proline in a polyproline type II (PPII) helix conformation, was developed. The strategy is based on a Ru-catalyzed ring-closing metathesis of a dipeptide (4) prepared by PyBOP coupling of cis-5-vinylproline tert-butylester (2) and trans-N-Boc-3-vinylproline (rac-3) followed by chromatographic diastereomer separation. Building block 2 was prepared from L-proline in six steps via electrochemical C5-methoxylation, cyanation and conversion of the nitrile into a vinyl substituent. Building block rac-3 was prepared in five steps exploiting a Cu-catalyzed 1,4-addition of vinyl-MgBr to a 2,3-dehydroproline derivative in the key step. In the course of the investigation subtle dependencies of protecting groups on the reactivity of the 2,3- and 2,5-disubstituted pyrrolidine derivatives were observed. The configuration and conformational preference of several intermediates were determined by X-ray crystallography. The developed synthesis allows the preparation of substantial amounts of 6, which will be used in the search for new small molecules for the modulation of protein-protein interactions involving proline-rich motifs (PRDs).

Concise, stereodivergent and highly stereoselective synthesis of cis- and trans-2-substituted 3-hydroxypiperidines - development of a phosphite-driven cyclodehydration.

Summary A concise (5 to 6 steps), stereodivergent, highly diastereoselective (dr up to >19:1 for both stereoisomers) and scalable synthesis (up to 14 g) of cis- and trans-2-substituted 3-piperidinols, a core motif in numerous bioactive compounds, is presented. This sequence allowed an efficient synthesis of the NK-1 inhibitor L-733,060 in 8 steps. Additionally, a cyclodehydration-realizing simple triethylphosphite as a substitute for triphenylphosphine is developed. Here the stoichiometric oxidized P(V)-byproduct (triethylphosphate) is easily removed during the work up through saponification overcoming separation difficulties usually associated to triphenylphosphine oxide.

A General Catalytic Method for Highly Cost- and Atom-Efficient Nucleophilic Substitutions

A general formamide-catalyzed protocol for the efficient transformation of alcohols into alkyl chlorides, which is promoted by substoichiometric amounts (down to 34 mol %) of inexpensive trichlorotriazine (TCT), is introduced. This is the first example of a TCT-mediated dihydroxychlorination of an OH-containing substrate (e.g., alcohols and carboxylic acids) in which all three chlorine atoms of TCT are transferred to the starting material. The consequently enhanced atom economy facilitates a significantly improved waste balance (E-factors down to 4), cost efficiency, and scalability (>50 g). Furthermore, the current procedure is distinguished by high levels of functional-group compatibility and stereoselectivity, as only weakly acidic cyanuric acid is released as exclusive byproduct. Finally, a one-pot protocol for the preparation of amines, azides, ethers, and sulfides enabled the synthesis of the drug rivastigmine with twofold SN 2 inversion, which demonstrates the high practical value of the presented method.